Multiplex digital nucleic acid quantitation using molecular barcodes Paul Rasmussen Sr. Manager of...

Multiplex digital nucleic acid quantitation using molecular barcodes

Paul Rasmussen

Sr. Manager of Emerging Markets and Consumable Programs

[email protected]

NanoString Confidential.1


Agenda

Platform Introduction

Chemistry Overview

Performance

Application extensions miRNA and CNVs

NanoString nCounter assay: Single reaction, up to 800 targets

QPCR

Microarrays/NGS

Sens

itivity

Multiplexing # of Transcripts

NanoString

3 NanoString Confidential.

1 10 100 1000 10,000

4

The nCounter platform facilitates powerful research

Over 100 papers have been published using the nCounter platform as of June-2012, at a rate more than doubling yearly

>20% are published in Science, Nature, Cell, or PNAS

Publications span most major disciplines in molecular biology Cancer, Immunology, Stem Cells, Systems Biology, Agriculture

Driven by performance with FFPE and remarkable precisionAccess previously unusable samples Observe biology not previously possible

Publication rate underestimates utilization of the platform by large pharma and industry.

Often less motivated to publish

2008 2009 2010 2011 20120

10

20

30

40

50

60

70

80

90

projected by EOY

pubs per year

The nCounter Analysis System: Two fully automated instruments

Fully Automated sample processing

Up to 800 genes per sample

12 samples processed in one cartridge

Up to 4 cartridges per day

Fully automated imaging and counting

Up to 6 cartridges (72 samples) per day

24 hour unattended processing

Simple data output

5

nCounter Prep Station

nCounter Digital Analyzer


Agenda


Chemistry Overview

Performance


Each Barcode Attached to an Individual RNA

Two Probe Assay

Target Specific Capture & Reporter Probes are created to bind to the mRNA transcript

Biotin

Target Specific Capture Probe

Target Specific Reporter Probe


Both probes must hybridize

Target Specific Capture & Reporter Probes are created to bind to the mRNA transcript



9

nCounter CodeSet

Pre-mixed sets of all probes and controls

0

100

200

300

400

500

600

0 20 40 60 80 100 120

Capture Probes

Reporter Probes

System Controls


Day 2AUTOMATED

The nCounter Assay: Three Simple Steps

5 minHANDS-ON

Day 2AUTOMATED

5 minHANDS-ON

Day 15 minHANDS-ON

Purify2


nCounter Prep Station

nCounter Digital Analyzer

Hybridize1

Flexible sample requirements

Only 4 pipetting steps

No amplification

800 hybridizations in single tube

Count3

Sensitive

Precise

Quantitative

Simple

Removeexcessreporters

Bindreporterto surface

Immobilize and align reporter

Image surface

HybridizeCodeSet to RNA

Count codes

nCounter Assay

mRNA Capture & Reporter Probes





Image surface


Count codes

nCounter Assay

Hybridized mRNA Excess Reporters





Image surface


Count codes

nCounter Assay

Hybridized Probes Bind to Cartridge

Surface of cartridge is coated with streptavidin





Image surface


Count codes

nCounter Assay

Immobilize and align reporter for image collecting and barcode counting





Image surface


Count codes

nCounter Assay

Image Surface

One coded reporter = 1 mRNA


Code Gene Countx 3

y 1

z 2

nCounter Assay

Codes are counted and tabulated




Image surface


Count codes



Simple read out of counts


Agenda


Chemistry Overview

Performance


The nCounter Assay: Very Reproducible

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000

R2 = 0.9999

Repl

icate

1 C

ount

sReproducibility of NanoString Assay Technical Replicates

Replicate 2 Counts

Data Courtesy of Dr. Roger Bumgarner


Superior Precision in Site-to-Site Reproducibility

Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastoma samples

Northcott P.E. et al., Acta Neuropathologica; November 16, 2011

“We present an assay based on NanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials.”

Site 1 Site 2

Site 3

R2 Site1 v Site 2 = 0.97R2 Site1 v Site 3 = 0.98

NanoString® Technologies | Confidential23

Very good cross platform performance: qPCR

Khan et al., 2011

Payton et al, High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples. J Clin Invest. June 2009

Very good cross platform performance: Affymetrix

PCA of signature on Affymetrix and NanoString

Payton et al, High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples. J Clin Invest. June 2009

Very good cross platform performance: RNA-Seq


Sun et al. Integrated analysis of Gene Expression, CpG Island Methylation, and Gene Copy Number in Breast Cancer Cells by Deep Sequencing. PLoSone, Feb, 2011


What samples are you using: flexible options

Total RNA and DNA (100ng/300ng/sample)

Amplified RNA from Small Amount of Sample

LCM and single cell (in progress)

Whole Cell Lysates

PaxGene Lysed Whole Blood

Total RNA and DNA Extracted from FFPE Samples

Crude Extracts from FFPE samples

Plasma, Serum and other Biofluids

Formalin fixation inhibits qPCR much more than the nCounter platform

Heart Frozen/FFPE

0

1

10

100

POLR2a LDHA GRPR C13orf23 NDUFV3

PCRnCounter

Fold

Dec

reas

e


Unparalleled Performance on FFPE Samples

mRNA Transcript Quantification in Archival Samples Using Multiplexed, Color-coded ProbesReis, P.P. et al., BMC Biotechnology; May 9, 2011

“… the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples.We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.”

nCounter® (r = 0.90) qPCR (r = 0.50)


Sample flexibility: Cell Lysate and Matched Total RNA

1 10 100 1000 100000

1

10

100

1000

10000

f(x) = 0.298104770543451 x + 7.194209916585R² = 0.976173007157184

f(x) = 0.630093248384163 x + 12.3939054285541R² = 0.977808883097132

f(x) = 1.30173923776692 x + 22.2432014725715R² = 0.972857263216265

10,000 cells vs 100ngLinear (10,000 cells vs 100ng)5,000 cells vs 100ngLinear (5,000 cells vs 100ng)2,500 cells vs 100ngLinear (2,500 cells vs 100ng)

Counts in 100ng RNA

Coun

ts in

Lys

ate

Measurements with crude whole cell lysates correlate extremely well with purified RNA


Flexibility of sample input Total RNA vs Lysate

Khan et al., 2011


PaxGene Blood Lysates vs. Purified RNA

-2 0 2 4 6 8 10 12 14 16-2

0

2

4

6

8

10

12

14

16

18

f(x) = 0.985428679843974 x + 0.451779490446374R² = 0.987902256477999f(x) = 0.976041231786931 x + 0.337161048855161R² = 0.983418605725623

Blood Lysate Prep 1Linear (Blood Lysate Prep 1)

log2 counts from total RNA purified from blood

log2

cou

nts

from

blo

od ly

sate

Measurements with unpurified PAXgene blood lysates correlate extremely well with purified RNA


Agenda


Chemistry Overview

Performance

Application extensions miRNA, and CNVs

34

nCounter miRNA Assays

Human miRNA Panel

Detects:800 human miRNAs3 nonhuman miRNAs (possible spike in controls)

Mouse miRNA Panel

Detects:578 mature mouse miRNAs33 mature murine-associated viral miRNAs

Rat miRNA Panel

Detects:423 mature rat miRNAs

All miRNA Panels

Sample Types Supported: RNA from Fresh/frozen tissue, FFPE, Blood, Cells

Sample Input Recommendation:100 ng purified total RNA

Linear Dynamic Range: 2106 counts

Hands on Time: <2 hours


Current challenges to miRNA detection

Short lengthoverall low Tmprohibits concurrent binding, e.g. nCounter dual probe

Highly Related Sequences

Large sequence diversity leads to large Tm spread even though length distribution is fairly small

40 50 60 70 80 900.00%

1.00%

2.00%

3.00%

4.00%

5.00%

6.00%

Human miRNA Tm Distribution

Tm

% H

uman

miR

NA

s

36

miRNA Sample Preparation Basics

miRNAs

37


miRNAs

Hybridize bridge oligoto each miRNA target

38


miRNAs

Bridge oligo specifically annealsto each miRNA target

Unique miRtag for each miRNA species

39


miRNA is covalently linked to miRtag via ligationmiRNAs

Bridge oligo specifically annealsto each miRNA target

Unique miRtag for each miRNA species


Excess bridges and tags are removed

40

Probe Architecture

Target Specific Capture & Reporter Probes bind to the chimaeric miRNA:miRtag molecule



41

Biotin


nCounter miRNA Analysis

Multiplexed target profiling of miRNA transcriptomes in a single reaction

Available for human, mouse, rat and drosophila

High level of sensitivity, specificity, precision, and linearity

nCounter®

miRNA AnalysisUnambiguous Discrimination of miRNAs with

Single Nucleotide Differences

Dynamic range of the miRNA assay

Dynamic range of the miRNA assay

hsa-miR-1 expression in different tissues

New miRNA assays for the nCoutner assay

A la carte miRNAsSelect from 20-50 miRNAs from our panels for focused profilingWorkflow identical to standard miRNA assayUsers specify housekeepers (stable miRNAs for normalization)

miRGE assayMixed mRNA and miRNA codesets10-30 miRNAs from our panelsUp to 200 mRNAsWorkflow similar to our standard miRNA assay


10 100 1000 1000010

100

1000

10000

R² = 0.988600727780784

GEx vs miRGE assay format probe comparison

Counts for GEx format probes

Cou

nts

for m

iRG

E fo

rmat

pro

bes


nCounter Copy Number Assay – Sample Preparation


Genomic DNA Fragment ds genomic DNA using 4 base cutter (Alu 1)

Average~ 500 bases

Perform nCounter

Hybridization

Denature DNA @ 95C

Variable copies of X chromosome

Feasibility initially demonstrated with cell lines containing 1, 2, 3, 4 and 5 X chromosomes

Requires fragmentation and denaturation of genomic DNA

The accuracy (measured vs. expected values) obtained with the nCounter system is extremely high



Calls agree with HapMap

One copy No copies

No copies

HapMap CNV nCounter CNV


Experiment: 100 HapMap samples + reference assayed (600ng digest/300ng hyb)

20 CNV regions with 3 probes analyzed.

Metric % of total Data points

Call rate 94.8% 1902/2006

Accuracy (concordance) 94.2% 1791/1902

Very good correlation with HapMap


The importance of Karyotyping….

Normal Female

HeLa

Molecules That CountTM

Paul [email protected]

Thank You!

Multiplex digital nucleic acid quantitation using molecular barcodes Paul Rasmussen Sr. Manager of...

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Transcript of Multiplex digital nucleic acid quantitation using molecular barcodes Paul Rasmussen Sr. Manager of...